Protective Effects And Mechanism Of Insulin On Brain Injury In Rats With Sepsis Induced By Lipopolysaccharide

ObjectiveUncoupling protein 2(UCP2)is an inner mitochondrial membrane protein that regulate energy metabolism and reduce reactive oxygen species production.Its neuroprotective effect has been widely proved in Parkinson Disease,cerebral ischemia,epilepsy and other neurological diseases.In sepsis,recent studies show that insulin not only can reduce blood glucose and regulate metabolism,but also can reduce ROS production,resist oxidative stress damage,anti-apoptosis and protect mitochondria.However,the mechanisms are still not very clear.It has been found that insulin may increase UCP2 expression,and reduce the production of ROS,which play a protective role against oxidative stress damage.Our research aims to investigate the protective effects of insulin on brain injury in rats with sepsis.And to investigate the expression of UCP2 in brain tissue of rats with sepsis,and the effect of insulin on the expression of UCP2,and to explore the possible roles of UCP2 in the protective effects of insulin.In addition,this study also attempt to provide new experimental and theoretical foundations for the clinical application of insulin treatment of sepsis.MethodsAfter the concentration of insulin was determined by preliminary experiment based on the blood glucose level and the survival rate of rats,fifty male Sprague-Dawley rats were randomly divided into normal control group(n= 10),sepsis group(n=20)and insulin treatment group(n=20).The rat sepsis model was established through an intraperitoneal injection of 15 mg/kg lipopolysaccharide(LPS)of gram-negative bacteria.The rats in the insulin treatment group received a lU/kg long-acting insulin injection(determined by preliminary experiment)subcutaneously 30 minutes before the injection of LPS and the rats in normal control group were given equivalent normal saline in the same way.Eight rats in each group were killed and cerebral cortex were harvested after the injection of LPS for 24 hours.The mitochondia of cerebral cortex were extracted for detecting the levels of ROS,MDA and the activity of SOD.Western blot was used to determine the protein expression of Bcl-2,Bax and Cleaved Caspase-9.TUNEL staining was used to detect neuronal apoptosis.Hematoxylin-Eosin(HE)staining was used to detect pathological changes of cerebral cortex.RT-PCR was used to detect the mRNA expression of UCP2.And UCP2 protein expression was detected by Western blot and immunohistochemistry.Results(1)The levels of mitochondrial ROS and MDA in sepsis group and insulin treatment group significantly increased when compared with normal control group,whereas SOD activity decreased,all P<0.05.Compared with sepsis group,the levels of mitochodrial ROS and MDA in insulin treatment group decreased,while SOD activity increased,all P<0.05.(2)Compared with normal control group,the neuronal apoptosis of cortex in both sepsis and insulin treatment group increased,and in sepsis group,the expression of Bcl-2 decreased,while the expression of Bax and Cleaved Caspase-9 increased,all P<0.05.Compared with sepsis group,insulin treatment decreased the neuronal apoptosis of cortex,and increased the Bcl-2 expression,but reduced Bax and Cleaved Caspase-9 expression,all P<0.05.(3)Compared with normal control group,obvious abnormal pathological changes was revealed by Hematoxylin and Eosin staining in cerebral cortex of rats in sepsis group and insulin treatment group,and the pathological changes was attenuated in insulin treatment group when compared with sepsis group.(4)Compared with normal control group,the mRNA and protein expression of UCP2 were elevated in sepsis group.Compared with sepsis group,insulin treatment increased the mRNA and protein expression of UCP2.Conclusion1.Subcutaneous injection of lU/kg long-acting insulin improves the survival rate of septic rats.2.The expression of UCP2 in brain tissue of rats with sepsis was elevated.3.Insulin upregulates the expression of UCP2 in brain tissue of rats with sepsis.4.Insulin play a protective role in the brain injury of rats with sepsis through reducing ROS production,alleviating mitochondrial oxidative stress,inhibiting the mitochondrial-initiated apoptotic pathway and reducing neuronal apoptosis.Insulin upregulates UCP2 expression in brain of septic rats to reduce ROS production and reduce neuronal apoptosis,which may play a role in the protective effects that mentioned above.